Multiplexed Measurement of Psychoactive Drug Selectivity Profiles

描述(由申请人提供)：这一第一阶段SBIR提案的主题是开发一个筛选系统，该系统量化单个样本中16个不同GPCR的候选精神活性药物的影响。许多内源性GPCR配体针对多个受体。特定的生物效应通常是通过“选择性非选择性”实现的，即一小部分受体被特定的配体不同程度地激活。同样，许多精神活性药物之所以有效，是因为它们针对多个GPCR。对于药物滥用等多组分疾病，能够在广泛的GPCR中筛选化合物的影响，并选择具有适当激活特征的药物，将有利于治疗干预的途径。相反，激活非预期目标的药物可能会产生灾难性的影响。这些化合物还将受益于能够评估它们在广泛的毒理学相关GPCRs中的专一性。为了识别抑制或激活多个感兴趣的受体的化合物，需要一个同时描述这些反应的系统。Primity开发了一种新的GPCR检测方法，适用于90%以上的GPCR，该方法将与Primity的蜂窝条形码平台(CellCode)相结合，能够在一次检测中同时筛选16个GPCR靶标。为了将这些技术结合在一起，GPCR检测将首先在高通量流式细胞仪筛选环境中进行性能优化。下一步，将产生16个表达不同GPCRs的细胞系，并对其进行分析性能优化。为了多重这些靶点，每个细胞系都将被用唯一的遗传识别符进行条形码编码，以便所有16个细胞系可以同时混合、冷冻、解冻和分析。作为最后的验证，将用特定的激动剂对混合物进行测试，以确保每个细胞系准确地报告测试激动剂(EC50)和拮抗剂(IC50)的组合和单独的药理。这些研究的完成将提供一种在高度多元化的系统中分析受体激活的独特方法，大大减少进行GPCR筛查的时间和成本，同时确保产生非常重要的数据。 公共卫生相关性：药物滥用的机制和后果跨越多个生物目标。这里描述的系统是一种在广泛的靶点上识别新疗法的效果的方法，以发现已知药物、精神活性物质的意想不到的活性，并确定草药疗法的成分，导致选择和开发更好的治疗化合物。

DESCRIPTION (provided by applicant): The subject of this Phase I SBIR proposal is the development of a screening system that quantifies the effects of candidate psychoactive drugs across 16 different GPCRs in a single sample. Many endogenous GPCR ligands target multiple receptors. Specific biological effects are often achieved through a "selective non-selectivity" where a small subset of receptors is activated to varying degrees by a specific ligand. Similarly, many psychoactive drugs are only effective because they target multiple GPCRs. For multi-component diseases such as drug abuse, the path to therapeutic intervention would benefit from being able to screen compounds for their effects across a wide range of GPCRs and select drugs with the proper activation profiles. Conversely, drugs that activate unintended targets can have disastrous effects. These compounds would also benefit from being able to assess their specificity across a wide range of toxicologically relevant GPCRs. In order to identify compounds that inhibit or activate multiple receptors of interest, a system is needed that profiles these responses simultaneously. Primity has developed a novel GPCR assay, applicable to greater than 90% of all GPCRs, that will be combined with Primity's cellular barcoding platform (CellCode) to enable the simultaneous screening 16 GPCR targets in a single assay well. In order to combine these techniques, the GPCR assay will first be optimized for performance in a high-throughput flow cytometry screening environment. Next, sixteen cell lines expressing different GPCRs will be generated and optimized for performance in the assay. To multiplex these targets, each cell line will be barcoded with a unique genetic identifier such that all sixteen cell lines can be mixed, frozen, thawed, and analyzed simultaneously. As a final validation, the mixture will be tested with specific agonists to ensure each cell line accurately reports the pharmacology of the test agonists (EC50) and antagonists (IC50) in combination and individually. The completion of these studies will provide a unique method of assaying receptor activation in a highly multiplexed system that dramatically reduces the time and costs of performing GPCR screens, yet ensures highly significant data generation. PUBLIC HEALTH RELEVANCE: The mechanisms and consequences of drug abuse cross multiple biological targets. The system described here is a method of identifying the effects of novel therapies across a broad range of targets to find unexpected activities of known drugs, psychoactive substances, and to define the components of herbal therapies, leading to the selection and development of better therapeutic compounds.